Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes

ABSTRACT Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice. Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

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Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

mBio ◽

10.1128/mbio.01795-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 39

Author(s):

Bang Shen ◽

Jeffrey S. Buguliskis ◽

Tobie D. Lee ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Gliding Motility ◽

Surface Proteins ◽

Host Cell Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Transmembrane Segments ◽

Rhomboid Proteases

ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

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Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01889-19 ◽

2020 ◽

Vol 64 (3) ◽

Cited By ~ 1

Author(s):

Jixu Li ◽

Huanping Guo ◽

Eloiza May Galon ◽

Yang Gao ◽

Seung-Hun Lee ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Drug Targets ◽

Protozoan Parasite ◽

Cell Types ◽

Lytic Cycle ◽

Type I ◽

Content Type ◽

Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

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A Toxoplasma gondii Ortholog of Plasmodium GAMA Contributes to Parasite Attachment and Cell Invasion

mSphere ◽

10.1128/msphere.00012-16 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 10

Author(s):

My-Hang Huynh ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Genetic Evidence ◽

Specific Role ◽

Host Cells ◽

Human Pathogen ◽

Content Type ◽

Apicomplexan Parasites ◽

Adhesive Proteins

ABSTRACT Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion. Toxoplasma gondii and its Plasmodium kin share a well-conserved invasion process, including sequential secretion of adhesive molecules for host cell attachment and invasion. However, only a few orthologs have been shown to be important for efficient invasion by both genera. Bioinformatic screening to uncover potential new players in invasion identified a previously unrecognized T. gondii ortholog of Plasmodium glycosylphosphatidylinositol-anchored micronemal antigen (TgGAMA). We show that TgGAMA localizes to the micronemes and is processed into several proteolytic products within the parasite prior to secretion onto the parasite surface during invasion. TgGAMA from parasite lysate bound to several different host cell types in vitro, suggesting a role in parasite attachment. Consistent with this function, tetracycline-regulatable TgGAMA and TgGAMA knockout strains showed significant reductions in host cell invasion at the attachment step, with no defects in any of the other stages of the parasite lytic cycle. Together, the results of this work reveal a new conserved component of the adhesive repertoire of apicomplexan parasites. IMPORTANCE Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion.

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Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

mBio ◽

10.1128/mbio.00754-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

Shruthi Krishnamurthy ◽

Bin Deng ◽

Roxana del Rio ◽

Kerry R. Buchholz ◽

Moritz Treeck ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Transmembrane Domain ◽

Critical Role ◽

Host Cells ◽

Receptor Protein ◽

Host Cell Invasion ◽

Cell Binding ◽

Apicomplexan Parasites

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T . gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE Nearly one-third of the world’s population is infected with the protozoan parasite Toxoplasma gondii , which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

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A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion

mBio ◽

10.1128/mbio.01510-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 12

Author(s):

Bradley I. Coleman ◽

Sudeshna Saha ◽

Seiko Sato ◽

Klemens Engelberg ◽

David J. P. Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Host Cells ◽

Model Organisms ◽

Host Cell Invasion ◽

Content Type ◽

Calcium Dependent ◽

Calcium Sensing ◽

Intracellular Ca

ABSTRACT Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion. IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite’s infection cycle.

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Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽

10.1128/msphere.00226-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 8

Author(s):

Jennifer Timm ◽

Maria Valente ◽

Daniel García-Caballero ◽

Keith S. Wilson ◽

Dolores González-Pacanowska

Keyword(s):

Homologous Recombination ◽

Host Cell ◽

Trypanosoma Brucei ◽

Cell Invasion ◽

Essential Amino Acids ◽

Host Cell Invasion ◽

Content Type ◽

Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

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Conditional Expression of Toxoplasma gondii Apical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

Molecular Biology of the Cell ◽

10.1091/mbc.e05-04-0281 ◽

2005 ◽

Vol 16 (9) ◽

pp. 4341-4349 ◽

Cited By ~ 179

Author(s):

Jeffrey Mital ◽

Markus Meissner ◽

Dominique Soldati ◽

Gary E. Ward

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Apical Membrane ◽

Conditional Knockout ◽

Host Cell Invasion ◽

Apical Membrane Antigen 1 ◽

Apicomplexan Parasites ◽

Apical Membrane Antigen ◽

Conditional Expression

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.

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Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture

mBio ◽

10.1128/mbio.00988-21 ◽

2021 ◽

Author(s):

Eden R. Ferreira ◽

Alexis Bonfim-Melo ◽

Barbara A. Burleigh ◽

Jaime A. Costales ◽

Kevin M. Tyler ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Invasion ◽

Lytic Cycle ◽

Host Cells ◽

Host Cell Invasion ◽

High Quality ◽

Content Type ◽

Membrane Rupture

Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite’s lytic cycle: how parasites egress and thereby sustain round after round of infection.

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A Conserved Apicomplexan Microneme Protein Contributes to Toxoplasma gondii Invasion and Virulence

Infection and Immunity ◽

10.1128/iai.01877-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4358-4368 ◽

Cited By ~ 22

Author(s):

My-Hang Huynh ◽

Martin J. Boulanger ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Recognition ◽

Epidermal Growth Factor Domain ◽

Content Type ◽

Apicomplexan Parasites ◽

Epidermal Growth ◽

The Many ◽

Host Cell Recognition

ABSTRACTThe obligate intracellular parasiteToxoplasma gondiicritically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress, and virulence. Although previous work established that the sporozoite protein with an altered thrombospondin repeat (SPATR) is a micronemal protein conserved in other apicomplexan parasites, includingPlasmodium,Neospora, andEimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study, we assess the contribution ofT. gondiiSPATR (TgSPATR) toT. gondiiinvasion by genetically ablating it and restoring its expression by genetic complementation. Δspatrparasites were ∼50% reduced in invasion compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, Δspatrparasites were significantly attenuated, with ∼20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether thePlasmodium falciparumSPATR ortholog (PfSPATR) could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of Δspatrparasites, because of an apparent failure in secretion. Overall, the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.

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New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

mBio ◽

10.1128/mbio.01628-14 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 5

Author(s):

Gregory M. Wier ◽

Erica M. McGreevy ◽

Mark J. Brown ◽

Jon P. Boyle

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Click Reaction ◽

Severe Disease ◽

Protozoan Parasite ◽

Trna Synthetase ◽

Content Type ◽

Fluorescent Tags

ABSTRACTToxoplasma gondiiis an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.IMPORTANCEStudying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins duringin vitrogrowth. It should also be applicable duringin vivoinfections and in other apicomplexan parasites, including Plasmodium spp.

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